Increased expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoforms in urinary exosomes in pre-eclampsia.

BACKGROUND: Glycolysis is altered in various kidney diseases, but little is known about glycolysis in pre-eclampsia, a multi-system disorder with major pathological effects on the kidney. Urinary exosomes provide a non-invasive alternative for studying changes in kidney metabolism. This study aims to characterise the expression and phosphorylation of isozymes of the key glycolytic regulatory protein, 6-phosphofructokinase-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), in urinary exosomes of subjects with pre-eclampsia (PE), compared to normotensive non-pregnant (NC) and normotensive pregnant (NP) controls. METHODS: A cross-sectional study of NC (n = 19), NP (n = 23) and PE (n = 29) subjects was performed. Exosomes were isolated from urine samples by differential ultracentrifugation, and then analyzed by Western blot and densitometry for expression of PFK-2/FBPase-2 isozymes (PFKFB2, PFKFB3 and PFKFB4) and phosphorylation of PFKFB2 at residues Ser483 and Ser466 and PFKFB3 at Ser461. RESULTS: PFKFB2 expression was increased 4.7-fold in PE compared to NP (p < 0.001). PFKFB2 phosphorylation at Ser483 was increased 2.6-fold in PE compared to NP (p = 0.002). Expression of phosphorylated PFKFB2/PFKFB3 at Ser466/Ser461 was increased in PE, being present in 77.4% (95% CI 59.9-88.9%) of PE and 8.3% (95% CI 1.2-27.0%) of NP samples (p < 0.001). PFKFB3 was more commonly expressed in PE, detected in 90.3% (95% CI 74.3-97.4%) of PE and 8.3% (95% CI 1.2-27.0%) of NP samples (p < 0.001). PFKFB4 had a 7.2-fold increase in expression in PE compared to NP (p < 0.001). No significant differences between NP and NC groups were observed. CONCLUSION: Regulatory proteins that increase glycolysis are increased in the urinary exosomes of subjects with pre-eclampsia, suggesting that renal glycolysis may be increased in this condition.

Limited capacity of proximal tubular proteolysis in mice with proteinuria.

Albuminuria is associated with the additional loss in the urine of small molecular weight proteins normally degraded by the proximal convoluted tubule (PCT), and competition for binding to the megalin/cubilin reuptake system has been considered the likely cause. We have previously reported that deficiency of the intrinsic lysosomal protein Limp-2 causes tubular proteinuria due to reduced fusion of endosomes with lysosomes in the PCT leading to inadequate proteolysis. To determine whether this mechanism also contributes to the tubular proteinuria induced by albumin overload in normal mice, wild-type (WT) mice received daily BSA injections intraperitoneally for 10 days, using untreated Limp-2(-/-) mice as positive controls for inadequate proteolysis. BSA overload induced significant urinary loss of megalin and cubilin ligands in WT mice. Tubular uptake of Alexa-conjugated BSA, administered by intravenous injection, was not reduced in the PCT of mice receiving intraperitoneal BSA. Expression of the tubular protein receptor megalin was also unchanged. There was a delay in proteolysis of reabsorbed proteins in WT mice receiving BSA, evidenced by an increased quantity of retinol-binding protein (RBP) in the kidney cortex, increased basal distribution of endocytosed RBP in cells of the PCT, and persistence of exogenous Alexa-conjugated BSA and RBP after injection. Upregulation of cathepsin L and normal fusion of lysosomes with endosomes were apparently not sufficient to maintain normal clearance of endocytosed proteins. The data suggest that in the presence of competition from albumin overload, reabsorption of filtered proteins is limited by the capacity of lysosomal degradation rather than receptor-mediated endocytosis.

Expression of NEDD4L and ENaC in Urinary Extracellular Vesicles in Pre-eclampsia.

OBJECTIVE: To assess changes in expression of renal epithelial sodium channel (ENaC) and NEDD4L, a ubiquitin ligase, in urinary extracellular vesicles (UEV) of pre-eclamptic women compared to normal pregnant controls. METHODS: Urine was collected from pre-eclamptic women (PE, n = 20) or during normal pregnancy (NP, n = 20). UEV were separated by differential ultracentrifugation. NEDD4L, α-ENaC and γ-ENaC were identified by immunoblotting. RESULTS: There was no difference in the expression of NEDD4L (p = 0.17) and α-ENaC (p = 0.10). PE subjects showed increased expression of γ-ENaC by 6.9-fold compared to NP (p < 0.0001). CONCLUSION: ENaC expression is upregulated in UEV of pre-eclamptic subjects but was not associated with changes in NEDD4L.

Expression of the transmembrane lysosomal protein SCARB2/Limp-2 in renin secretory granules controls renin release.

BACKGROUND/AIMS: Renin processing and storage is believed to occur in lysosome-like structures in the afferent arteriole. SCARB2/Limp-2 is a transmembrane lysosomal protein responsible for the intracellular trafficking of ß-glucocerebrosidase. This study aimed to confirm the expression of SCARB2/Limp-2 in renin secretory granules, and explore its role in renin processing and secretion. METHODS: Co-localisation studies of (pro)renin with lysosomal membrane proteins, SCARB2/Limp-2, LAMP-1 and LAMP-2, were performed in mouse and human kidney sections. Intrarenal expression and secretion of (pro)renin in wild-type (WT) and Limp-2(-/-) mice were compared with and without stimulation. RESULTS: SCARB2/Limp-2, LAMP-1 and LAMP-2 co-localised with (pro)- renin in mouse and human kidney. Plasma renin concentration was increased in Limp-2(-/-) mice when compared to WT littermates. No change in (pro)renin expression, however, was observed in Limp-2(-/-) mouse kidney cortex by immunofluorescence microscopy, Western blotting, quantitative RT-PCR or the ultrastructural appearance of renin secretory granules. Acute stimulation of renin release by isoprenaline or hydralazine was similar in WT and Limp-2(-/-) mice. Following chronic salt restriction, however, immunofluorescence microscopy showed less (pro)renin expressed in Limp-2(-/-) compared with WT mouse kidneys, and there was significantly less prorenin but not renin by Western blotting in Limp-2(-/-) mouse kidney cortex, despite no difference in circulating renin levels. CONCLUSION: Renin secretory granules possess integral lysosomal proteins, confirming that they are indeed modified lysosomes. Limp-2 deficiency leads to a minor increase in circulating renin. Limp-2, however, is not required for acute or chronic stimulation of renin release.

Tubular proteinuria in mice and humans lacking the intrinsic lysosomal protein SCARB2/Limp-2.

Deficiency of the intrinsic lysosomal protein human scavenger receptor class B, member 2 (SCARB2; Limp-2 in mice) causes collapsing focal and segmental glomerular sclerosis (FSGS) and myoclonic epilepsy in humans, but patients with no apparent kidney damage have recently been described. We now demonstrate that these patients can develop tubular proteinuria. To determine the mechanism, mice deficient in Limp-2, the murine homolog of SCARB2, were studied. Most low-molecular-weight proteins filtered by the glomerulus are removed in the proximal convoluted tubule (PCT) by megalin/cubilin-dependent receptor-mediated endocytosis. Expression of megalin and cubilin was unchanged in Limp-2(-/-) mice, however, and the initial uptake of injected Alexa Fluor 555-conjugated bovine serum albumin (Alexa-BSA) was similar to wild-type mice, indicating that megalin/cubilin-dependent, receptor-mediated endocytosis was unaffected. There was a defect in proteolysis of reabsorbed proteins in the Limp-2(-/-) mice, demonstrated by the persistence of Alexa-BSA in the PCT compared with controls. This was associated with the failure of the lysosomal protease cathepsin B to colocalize with Alexa-BSA and endogenous retinol-binding protein in kidneys from Limp-2(-/-) mice. The data suggest that tubular proteinuria in Limp-2(-/-) mice is due to failure of endosomes containing reabsorbed proteins to fuse with lysosomes in the proximal tubule of the kidney. Failure of proteolysis is a novel mechanism for tubular proteinuria.

SCARB2 mutations in progressive myoclonus epilepsy (PME) without renal failure.

OBJECTIVE: Mutations in SCARB2 were recently described as causing action myoclonus renal failure syndrome (AMRF). We hypothesized that mutations in SCARB2 might account for unsolved cases of progressive myoclonus epilepsy (PME) without renal impairment, especially those resembling Unverricht-Lundborg disease (ULD). Additionally, we searched for mutations in the PRICKLE1 gene, newly recognized as a cause of PME mimicking ULD. METHODS: We reviewed cases of PME referred for diagnosis over two decades in which a molecular diagnosis had not been reached. Patients were classified according to age of onset, clinical pattern, and associated neurological signs into "ULD-like" and "not ULD-like." After exclusion of mutations in cystatin B (CSTB), DNA was examined for sequence variation in SCARB2 and PRICKLE1. RESULTS: Of 71 cases evaluated, 41 were "ULD-like" and five had SCARB2 mutations. None of 30 "not ULD-like" cases were positive. The five patients with SCARB2 mutations had onset between 14 and 26 years of age, with no evidence of renal failure during 5.5 to 15 years of follow-up; four were followed until death. One living patient had slight proteinuria. A subset of 25 cases were sequenced for PRICKLE1 and no mutations were found. INTERPRETATION: Mutations in SCARB2 are an important cause of hitherto unsolved cases of PME resembling ULD at onset. SCARB2 should be evaluated even in the absence of renal involvement. Onset is in teenage or young adult life. Molecular diagnosis is important for counseling the patient and family, particularly as the prognosis is worse than classical ULD.

Diabetes and higher HbA1c levels are independently associated with adverse renal outcomes in inpatients following multiple hospital admissions.

OBJECTIVE: To assess the association between glycaemic status prior to the first hospital presentation with developing adverse renal outcomes overtime in patients with multiple hospital re-admissions. DESIGN: A prospective observational cohort study. PARTICIPANTS: All inpatients aged ≥54 years admitted between 2013 and 16 to a tertiary hospital. MAIN OUTCOMES: We prospectively measured HbA1c levels in all inpatients aged ≥54 years admitted between 2013 and 16. Diabetes was defined as prior documented diagnosis of diabetes and/or HbA1c ≥6.5% (47·5 mmol/L). Included patients had ≥ two admissions (at least 90 days apart), baseline estimated glomerular filtration rate (eGFR) >30 ml/min/1·73m2 and no history of renal replacement therapy. We assessed several renal outcomes: (a) 50% decline in eGFR; (b) rapid decline in renal function (eGFR decline >5 mL/min/1·73m2/year) and (c) final eGFR<30 ml/min/1·73m2. RESULTS: Of 4126 inpatients with a median follow-up of 465 days (254, 740), 26% had diabetes. The presence of diabetes was associated with higher odds of (a) 50% decline in eGFR (OR = 1·42;95% CI:1·18-1·70;p < 0·001); (b) rapid decline in renal function (OR = 1·40;95%CI:1·20-1·63;p < 0·001), and (c) reaching eGFR<30 ml/min/1.73m2 (OR = 1·25;95%CI:1·03-1·53;p < 0·05). Every 1% (11 mmol/L) increase in baseline HbA1c was associated with significantly greater odds of (a) >50% decline in eGFR (OR = 1·07;95% CI:1·01-1·4;p < 0·05) and (b) rapid decline in renal function (OR = 1·11;95% CI:1·05-1·18;p < 0·001). CONCLUSIONS: In patients with ≥two admissions, the presence of diabetes and higher HbA1c levels were strongly and independently associated with adverse renal outcomes at follow up. Such patients are at high risk of relatively rapid deterioration in renal function and a logical target for structured preventive interventions.

Obesity and hypertension have differing oxidant handling molecular pathways in age-related chronic kidney disease.

Chronic kidney disease (CKD) in ageing is a burden on health systems worldwide. Rat models of age-related CKD linked with obesity and hypertension were used to investigate alterations in oxidant handling and energy metabolism to identify gene targets or markers for age-related CKD. Young adult (3 months) and old (21-24 months) spontaneously-hypertensive (SHR), normotensive Wistar-Kyoto (WKY) and Wistar rats (normotensive, obese in ageing) were compared for renal functional and physiological parameters, renal fibrosis and inflammation, oxidative stress (hemeoxygenase-1/HO-1), apoptosis and cell injury (including Bax:Bcl-2), phosphorylated and non-phosphorylated forms of oxidant and energy sensing proteins (p66Shc, AMPK), signal transduction proteins (ERK1/2, PKB), and transcription factors (NF-kappaB, FoxO1). All old rats were normoglycemic. Renal fibrosis, tubular epithelial apoptosis, interstitial macrophages and myofibroblasts (all p<0.05), p66Shc/phospho-p66 (p<0.05), Bax/Bcl-2 ratio (p<0.05) and NF-kappaB expression (p<0.01) were highest in old obese Wistars. Expression of phospho-FoxO/FoxO was elevated in old Wistars (p<0.001) and WKYs (p<0.01). SHRs had high levels in young and old rats. Expression of PKB, phospho-PKB, ERK1/2 and phospho-ERK1/2 were significantly elevated in all aged animals. These results suggest that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related CKD.

Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Streptococcus salivarius K12.

A powder preparation of the oral probiotic Streptococcus salivarius K12 has been given to 19 young otitis media-prone children following a 3-day course of amoxicillin administered as a preliminary to ventilation tube placement. In two subjects, the use of strain K12 appeared to effect the expansion of an indigenous population of inhibitory S. salivarius. In other children, strain K12 colonisation extended beyond the oral cavity to also include the nasopharynx or adenoid tissue. The relatively low proportion (33%) of subjects that colonised was attributed to failure of the amoxicillin pre-treatment to sufficiently reduce the indigenous S. salivarius populations prior to dosing with strain K12 powder.

Compliance of acute and long stay geriatric patients with nutritional supplementation.

BACKGROUND: Nutritional supplements are prescribed to improve nutritional status, and reduce hospital stays in manourised hospital patients. Clinical benefits are dependant on compliance, the level of which remains unclear. AIMS: To assess compliance levels with oral nutritional supplementation and determine methods to improve compliance. METHODS: Compliance was observed over 10 days by measuring total supplements prescribed and weighing wastage remaining after use. Areas for improvement were identified and implemented for 6 months. Specifically, a distinct supplement administration round was established and those patients requiring assistance with supplement consumption were identified with signage above their beds. Compliance was re-assessed in a sub sample of patients. RESULTS: Thirty seven elderly patients (mean age 85 years; 57% female) prescribed nutritional supplements were studied. Mean compliance was significantly greater in males than females (85.7% vs 74%) and acute wards compared to longstay (89.5% vs. 74.2 Compliance with supplements was significantly greater following intervention (mean 74.2% vs. 93%, p < 0.0001). CONCLUSION: Compliance with nutritional supplementation is variable among institutionalized geriatric patients. Timing of supplementation dispensation and improving staff vigilance can positively affect compliance.

Complex organisation of the 5'-end of the human glycine tRNA synthetase gene.

Glycine tRNA synthetase (glyRS) catalyses the addition of the amino acid glycine to its cognate tRNA molecules. In the silk moth worm Bombyx mori, this gene is subject to complex transcriptional regulation because of the predominance of glycine in silk. In vertebrates, glycine is a major constituent of collagen but there have been no studies of glyRS regulation. In this study we have isolated and mapped a genomic clone containing the 5'-end of glyRS. Primer extension studies identified only one transcriptional start point (TSP) in three different cell lines. Expression of the transcript identified may be regulated translationally because it contains five potential initiation codons, three of which are in good context for initiation. The most 3' of the potential initiation codons has previously been predicted to be the initiating codon for cytoplasmic glyRS. Two of the upstream codons are in-frame with this codon, and both are predicted to extend the N-terminus of glyRS to include a mitochondrial targeting sequence. Sequencing of genomic DNA surrounding the TSP showed features common to the promoters of housekeeping genes, as well as a canonical TATA box at the unusual position of +9. Surprisingly, promoter activity in vitro was not specified by a 1.9 kb genomic fragment containing the TSP and TATA box, but by a contiguous 0.4 kb fragment immediately downstream. These studies suggest that the transcription of glyRS from a single start point requires downstream promoter elements.

AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and non-catalytic, beta and gamma (38 kDa) subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and we find that rat liver AMPK-alpha2 isoform catalytic subunit is associated with beta1 and gamma1 and not with beta2 or gamma2 subunit isoforms. The beta1 and gamma1 isoforms are also subunits of the alpha1 isoform. The sequence of cloned human AMPK-beta1 is 95% identical in amino acid sequence with rat beta1. Human chromosomal localizations were determined for AMPK-alpha1 (5p11-p14), AMPK-beta1 (12q24.1-24.3) and AMPK-gamma1 (12q12-q14), respectively.

AMP-activated protein kinase phosphorylation of endothelial NO synthase.

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.

The influence of repeated transfusions and cyclosporine on secondary alloantibody responses in inbred rats.

The influence of cyclosporine (CsA) on secondary and established alloantibody responses was evaluated in inbred Lewis rats and (AO x PVG)F1 hybrid rats. Lewis rats received weekly transfusions of DA whole blood for 8 weeks either with or without cyclosporine (15 mg/kg/day) after sensitization with DA splenocytes. Hybrid rats received only CsA (10 mg/kg/day) after similar sensitization. Administration of CsA did not affect the spontaneous decline in alloantibody titers against class I (RT1A) antigens, but it was associated with a significantly reduced response to class II (RT1B) antigens at the end of the study. CsA prevented maintenance of high alloantibody titers to RT1A antigens in Lewis rats transfused repeatedly following sensitization. IgG alloantibody subclass responses were also altered by CsA with significant reduction in titers of IgG1, 2a, and 2b against RT1A antigens in rats transfused repeatedly; CsA did not, however, suppress IgG2c alloantibody levels in these animals. Responses to public RT1A antigens disappeared in most animals irrespective of their treatment group, whereas those to private and public RT1B antigens persisted unless CsA was administered. The results suggest that, contrary to results obtained with other antigens, CsA does influence secondary alloantibody responses. CsA may thus prove of value in highly sensitized dialysis patients who require further blood transfusions.

A human lymphocytotoxic autoantibody is encoded by antibody variable region genes in their germ line configuration.

Lymphocytotoxic autoantibodies are commonly found in sera from kidney dialysis patients, where they interfere with interpretation of the crossmatch test. We have produced a monoclonal lymphocytotoxic autoantibody by EBV transformation of PBLs from a dialysis patient, followed by fusion of the transformed cells with a heteromyeloma. The autoantibody derived, called FWE, was IgM kappa class and its pattern of reactivity against T and B lymphocytes, cells from patients with chronic lymphocytic leukemia and K562, was similar to that described for lymphocytotoxic autoantibodies found in sera. It was absorbed by fetal but not adult human erythrocytes, suggesting the antigenic determinant might be the blood group antigen i. cDNA encoding the variable domain of FWE was amplified by polymerase chain reaction, cloned into the vector M13 mp18, and sequenced. The variable region of the kappa light chain (V kappa) was 98.8% identical over a 260-bp stretch with a known germ line sequence and the junctional (J kappa) region was identical over 16 bp with the germ line sequence J kappa 2. The variable region of the heavy chain (VH) was 99.3% identical over a 268-bp overlap with the germ line gene VH4.21, a member of the VHIV family, and the junctional region of the heavy chain (JH) was identical with the germ line JH gene JH5 over 46 bp, with a truncated 5' end. The diversity region was not identified. These data suggest that the genes required to produce human lymphocytotoxic autoantibodies are encoded within the germ line and, therefore, that all dialysis patients may be able to produce them under certain circumstances.

Immune responses to noninherited maternal RT1A antigens in inbred rats.

Humoral responses to non-inherited maternal class I antigens (class I NIMAs) were assessed in 3 groups of inbred rats expressing the RT1u phenotype. Group 1 consisted of the progeny of (AO X DA)F1 X PVG matings; their haplotype was RT1u/c and their non-inherited maternal haplotype RT1a. Group 2 were the progeny of (AO X LEW)F1 X PVG matings the haplotype of which was also RT1u/c, but their non-inherited maternal haplotype was RT1l. Group 3 comprised 8 (AO X PVG)F1 (RT1u/c) hybrids. All rats received 2 intravenous blood transfusions (0.5 ml) from male DA (RT1Aa) donors on days 0 and 7. They were bled at weekly intervals for 6 weeks and again at 20 weeks after the first transfusion. Alloantibody responses to RT1Aa were assessed by an indirect hemagglutination assay (IHA)* and by a 51Chromium-release complement-dependent red cell cytotoxicity assay. All groups exhibited vigorous anti-class I antibody responses to the DA transfusions. No significant differences were detected, however, in antibody titers between the groups either by IHA or CDC or in the rates of decay of antibody titers up to week 20. In addition no blocking activity was found in sera obtained on day 0 from group 1 animals and tested for antiidiotypic antibody activity to cytotoxic anti-RT1Aa antibodies. In order to assess whether suppressor activity had been activated by the initial transfusions, in the animals in which class I NIMA was RT1Aa, all groups were rechallenged with a DA transfusion at week 20. All animals exhibited vigorous anamnestic responses to this challenge and no significant differences were detected between groups. In order to determine whether cellular tolerance to the noninherited maternal haplotype was present in group 1 animals, proliferative responses were assessed by one-way mixed lymphocyte cultures, using DA lymph node stimulator cells. No significant differences were detected in proliferative or kinetic responses between lymph node cells from rats the noninherited maternal haplotype of which was RT1a or from naive (AO X PVG)F1 hybrids. Peak proliferative responses to DA cells in rats the noninherited maternal haplotype of which was RT1l were similar, but maximal on day 4 as opposed to day 3. Hence in these inbred rat strains no evidence of humoral tolerance to class I NIMAs was detected. In addition there was no evidence of cellular tolerance to the noninherited maternal MHC haplotype.

The alloantibody response to semiallogeneic pregnancy in the rat. I. Alloantibodies in sera and placental eluates directed to RT1A antigens.

Maternal alloantibodies to paternal cells were monitored by cellular ELISA, the indirect hemagglutination and erythrocyte antibody rosette inhibition assays in sera and placental eluates from primigravid and multigravid inbred rats. In primigravid animals, antibodies in sera were routinely detected only by the indirect hemagglutination assay and were of low titer; weak antibody activity was detectable only by indirect hemagglutination in 1 of the 8 placental eluates assayed from these animals. Alloantibodies in high titer were present in sera and placental eluates from multigravid rats and were found to be directed predominantly to the RT1A (class I MHC) antigens of the paternal strain. These data provide no support for the hypothesis that the difficulty in detecting maternal antibodies during a 1st pregnancy is due to their preferential binding to antigenic determinants expressed on the placenta.

The effect of cyclosporine on humoral and cellular alloreactivity to allogeneic pregnancy in rats.

We have evaluated the effect of a therapeutic dose (10 mg/kg/day) of the immunosuppressant cyclosporine (CsA) on humoral and cell-mediated immunity in rats during an allogeneic first pregnancy. Virgin female Lewis rats mated with DA males and treated with CsA vehicle produced a humoral response, as measured by both the erythrocyte rosette inhibition (EAI) and indirect hemagglutination assays. The capacity of Lewis splenocytes to mediate a graft-versus-host (GVH) reaction in six-week old F1 (Lewis X DA) hybrid rats was unaffected by either pregnancy or CsA. In mothers treated with CsA, however, no antibody production was detected, and a significant reduction in GVH reactivity was observed using their cells. This reduction was specific for the paternal strain. When compared with vehicle-treated primiparas, suppression of the immune response by CsA had no effect on either the number or viability of fetuses present in utero at day 20. These data suggest that antipaternal antibodies may not be essential to protect the fetus when there is concomitant suppression of the capacity of the mother's T cells to express a cell-mediated antipaternal response.

Alloantibody and transferable suppressor activity induced by cyclosporine and blood transfusions in the rat.

The effect of cyclosporine on the alloantibody response to blood transfusion was investigated in inbred strains of rats by IHA and CELISA; recipient animals differed from the donors at the class I (RT1A) or both class I and class II (RT1B) antigens of the major histocompatibility complex. Alloantibody titers stimulated in high responder PVGu/c animals by blood transfusions were attenuated by cyclosporine; this effect was not demonstrated in low responder PVGc rats, as alloantibody titers decreased after further blood transfusions whether or not cyclosporine was given. Cyclosporine not only reduced the initial IgM response but suppressed the subsequent production of IgG. Splenocytes from rats receiving cyclosporine and blood transfusions from donors that differed from the recipients at the class I antigen were effective in suppressing the subsequent antibody response to blood transfusion. When blood transfusions from donors which differed from the recipients at both class I and class II antigenic loci were given after splenocyte transfer, a greater degree of immunosuppression was detected than if the transfusion donor differed only at the class I locus. These data suggest that the sensitization produced by blood transfusions and the persistence or decline of the alloantibody response depend upon the responder status of the recipient. Blood transfusions given with cyclosporine are capable of inducing suppressor activity that is transferable in spleen homogenates. Subsequent alloantibody responses are influenced by the class I and class II disparities of the donor and recipient animals. If these results can be extrapolated to clinical practice, cyclosporine should be given with pretransplant blood transfusions to prevent sensitization, and the transfusion donor should differ from the recipient at both class I and class II antigenic loci.

The alloantibody response to semiallogeneic pregnancy in the rat. II. Antibodies in serum directed to multiple epitopes on conventional RT1A antigens.

Evidence for a unique class I MHC antigen (termed Pa), which is believed to be expressed on rat trophoblast during pregnancy and which stimulates alloantibody formation with unusual interstrain cross-reactivity, has been examined in inbred rats. The previously reported pattern of crossreactivity was confirmed but was not unique to antisera produced by pregnancy. Antibody blocking studies using biotinylated rat monoclonal antibodies to distinct epitopes on RT1Aa antigens suggested that antibodies present in pregnancy sera, especially from multiparous rats, reacted with several epitopes on these molecules. Moreover, a rat monoclonal antibody, 381- 1E10, directed against the putative Pa epitope was shown by synergistic lysis and cold antibody competition to be directed to the immunodominant S epitope on RT1Aa. These data argue against the existence of a distinct Pa antigen or epitope detected by pregnancy sera.